Project Summary/Abstract: At the level of individual cells, expression of genes is inherently stochastic across organisms ranging from prokaryotes to human cells. Stochastic expression manifests as cell-to-cell variation in gene product levels even among isogenic cells, and this variation significantly affects biological functions and phenotype. How cells ensure precision in the timing of key intracellular events in the face of of stochastic expression is an intriguing fundamental problem. One simple model for studying event timing is the phage ?'s lysis system, where lysis of the infected E. coli cell occurs when a protein, holin, reaches a critical threshold concentration in the cell membrane. Intriguingly, preliminary data reveals precision in timing: individual cells lyse at an optimally scheduled time with minimal statistical fluctuations in timing. The key objective of this proposal is to use ?'s lysis system to uncover regulatory mechanisms essential for buffering noise in timing at the single-cell level. Mathematically, noise in the event timing is investigated using the first-passage time framework, where an event is triggered when a stochastic process (holin level) hits a threshold for the first time. Novel analytical calculations of the first-passage time will be performed for stochastic models of gene expression and regulation of varying complexities. Combining theoretical results with single-cell lysis time measurements in both wild-type and mutant phages, the mechanisms controlling stochasticity in the timing of intracellular events will be characterized. In addition, we will use combination of mathematical models and experiments to determine how stochasticity in lysis times drives intercellular variations in the ? progeny count per cell. The first-passage time framework developed here is quite general as timing of diverse cellular processes depends on regulatory molecules reaching critical threshold levels. Identification of regulatory motifs that buffer randomness in the timing of intracellular events has consequences for cell-cycle control and development, where precision is required for proper system functioning. Quantitative characterization of ?'s lysis system is also critical for emerging medical applications such as using holin proteins for targeting cancer cells and pathogenic bacteria.